U.S. patent application number 11/803172 was filed with the patent office on 2007-10-25 for assays, antibodies, and standards for detection of oxidized and mda-modified low density lipoproteins.
This patent application is currently assigned to LEUVEN RESEARCH & DEVELOPMENT VZW. Invention is credited to Desire Jose Collen, Paul Noel Holvoet.
Application Number | 20070249006 11/803172 |
Document ID | / |
Family ID | 32116213 |
Filed Date | 2007-10-25 |
United States Patent
Application |
20070249006 |
Kind Code |
A1 |
Holvoet; Paul Noel ; et
al. |
October 25, 2007 |
Assays, antibodies, and standards for detection of oxidized and
MDA-modified low density lipoproteins
Abstract
Immunoassays for malondialdehyde-modified low density
lipoprotein (MDA-modified LDL) and oxidized low density lipoprotein
(OxLDL), monoclonal antibodies (and the cell lines for them) for
use in the assays, and a storage-stable standard (which may be used
as a calibrator and/or control) are disclosed. MDA-modified LDL and
OxLDL are implicated in atherosclerosis and its etiology.
Inventors: |
Holvoet; Paul Noel;
(Kessel-Lo, BE) ; Collen; Desire Jose; (London,
GB) |
Correspondence
Address: |
Stephen P. Gilbert, Esq.;BRYAN CAVE LLP
1290 Avenue of the America
New York
NY
10104-3300
US
|
Assignee: |
LEUVEN RESEARCH & DEVELOPMENT
VZW
|
Family ID: |
32116213 |
Appl. No.: |
11/803172 |
Filed: |
May 11, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10802709 |
Mar 17, 2004 |
7229776 |
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11803172 |
May 11, 2007 |
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09446259 |
Dec 20, 1999 |
6727102 |
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PCT/EP97/03493 |
Jul 1, 1997 |
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10802709 |
Mar 17, 2004 |
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Current U.S.
Class: |
435/7.94 ;
436/13 |
Current CPC
Class: |
G01N 33/96 20130101;
G01N 2800/044 20130101; G01N 33/92 20130101; Y10T 436/104165
20150115; C07K 16/18 20130101; C07K 2317/92 20130101; G01N 2800/323
20130101 |
Class at
Publication: |
435/007.94 ;
436/013 |
International
Class: |
G01N 33/53 20060101
G01N033/53 |
Foreign Application Data
Date |
Code |
Application Number |
Jun 20, 1997 |
EP |
PCT/EP97/03287 |
Claims
1-55. (canceled)
56. An immunological sandwich assay for the detection and/or
quantification of human MDA-modified LDL (malondialdehyde-modified
low density lipoprotein) in a sample derived from the body fluids
or tissues of a human being in which assay a first antibody that
has a high affinity for human MDA-modified LDL is bound to a
substrate, said assay comprising: (a) contacting the sample with
the substrate having bound to it the first antibody under binding
conditions so that at least some of any human MDA-modified LDL in
the sample will bind to the first antibody; (b) thereafter removing
unbound sample from the substrate; (c) thereafter contacting the
substrate with a second antibody that has a high affinity for human
MDA-modified LDL; and (d) thereafter visualizing and/or quantifying
the MDA-modified LDL that was present in the sample; wherein the
MDA-modified LDL for which the first antibody and the second
antibody have high affinity contains at least 60 substituted lysine
moieties per apo B-100 (apolipoprotein B-100) moiety.
57. The assay of claim 56 in which the first antibody also has high
affinity for human OxLDL (oxidized low density lipoprotein).
58. The assay of claim 56 in which the first antibody has low
affinity for human native LDL (low density lipoprotein).
59. The assay of claim 56 in which the first antibody has low
affinity for human OxLDL.
60. The assay of claim 56 in which the second antibody has high
affinity for human native LDL.
61. The assay of claim 56 in which the first antibody is the
monoclonal antibody mAb-4E6 produced by hybridoma Hyb4E6 deposited
at the BCCM (Belgian Coordinated Collections of Microorganisms)
under deposit accession number LMBP 1660 CB on Apr. 24, 1997.
62. The assay of claim 56 in which the first antibody is the
monoclonal antibody mAb-1H11 produced by hybridoma Hyb1H11
deposited at the BCCM under deposit accession number LMBP 1659 CB
on Apr. 24, 1997.
63. The assay of claim 56 in which the second antibody is the
monoclonal antibody mAb-8A2 produced by hybridoma Hyb8A2 deposited
at the BCCM under deposit accession number LMBP 1661 CB on Apr. 24,
1997.
64. A method of standardizing an assay for human MDA-modified LDL
and human OxLDL by using as a calibrator or as a control a stable
standard containing MDA-modified LDL whose extent of substitution
of its lysine moieties will remain essentially constant over normal
periods of time during normal storage for biological materials, the
MDA-modified LDL of said standard being made by contacting
malondialdehyde with LDL at a predetermined molar ratio of
malondialdehyde to the apo B-100 moiety of the LDL, the standard
containing an agent that reduces the ability of any metal ions
present to catalyze oxidation of the LDL and/or an
anti-oxidant.
65. The method of claim 64 wherein the standard contains both an
agent that reduces the ability of any metal ions present to
catalyze oxidation of the LDL and an anti-oxidant.
66. The method of claim 64 wherein the agent that reduces the
ability of any metal ions present to catalyze oxidation of the LDL
is a chelating agent.
67. The method of claim 64 wherein the standard further comprises a
physiological fluid.
68. The method of claim 64 wherein the standard further comprises
at least one anti-platelet coagulation compound and/or
anti-coagulant.
69. The method of claim 64 wherein the standard is used as a
calibrator.
70. The method of claim 64 wherein the standard is used as a
control.
71. A kit for conducting a sandwich assay for the determination of
human OxLDL or human MDA-modified LDL or both in a sample derived
from the body fluids or tissues of a human being, said kit
comprising: (a) a substrate on which is bound a first antibody that
has high affinity for human OxLDL or human MDA-modified LDL or
both, the OxLDL and MDA-modified LDL each having at least 60
substituted lysine moieties per apo B-100 moiety, and (b) a labeled
antibody having a high affinity for human OxLDL that becomes bound
to the first antibody during the assay or for human MDA-modified
LDL that becomes bound to the first antibody during the assay or
for both that become bound to the first antibody during the
assay.
72. The kit of claim 71 further comprising a reactive substance for
reaction with the labeled antibody to give an indication of the
presence of the labeled antibody.
73. The kit of claim 72 wherein the reactive substance comprises an
enzyme.
74. The kit of claim 71 further comprising the calibrator of claim
69.
75. The kit of claim 71 further comprising the control of claim 70.
Description
BACKGROUND
[0001] The present invention relates to assays, antibodies
(particularly monoclonal antibodies), and standards for detection
(i.e., determination of the presence and/or quantitation of the
amount) of oxidized low density lipoprotein (OxLDL) and
malondialdehyde-modified low density lipoprotein (MDA-modified LDL)
in samples, the samples typically being derived from body fluids or
tissues.
[0002] Lipoproteins are multicomponent complexes of protein and
lipids. Each type of lipoprotein has a characteristic molecular
weight, size, chemical composition, density, and physical role. The
protein and lipid are held together by noncovalent forces.
[0003] Lipoproteins can be classified on the basis of their density
as determined by ultracentrifugation. Thus, four classes of
lipoproteins can be distinguished: High Density Lipoproteins (HDL),
Intermediate Density Lipoproteins (IDL), Low Density Lipoproteins
(LDL), and Very Low Density Lipoproteins (VLDL).
[0004] The purified protein components of a lipoprotein particle
are called apolipoproteins (apo). Each type of lipoprotein has a
characteristic apolipoprotein composition. In LDL the prominent
apolipoprotein protein is apo B-100. Apo B-100 is one of the
longest single chain polypeptides known and consists of 4536 amino
acids. Of these amino acids the lysine residues or moieties (there
are 356 such lysine residues or moieties) can be substituted or
modified by aldehydes (e.g., malondialdehyde). Oxidation of the
lipids in LDL (whether in vitro, e.g., by copper-induced oxidation,
or whether in vivo) results in the generation of reactive
aldehydes, which can then interact with the lysine residues or
moieties of apo B-100. The outcome of this lysine substitution or
modification is that the resulting OxLDL, which is also
MDA-modified LDL, is no longer recognized by the LDL receptor at
the surface of fibroblasts but by scavenger receptors at the
surface of macrophages. At least 60 out of the 356 lysines (or
lysine residues or moieties) of apo B-100 have to be substituted in
order to be recognized by the scavenger receptors (see document
number 1 of the documents listed near the end of this application,
all of which documents are hereby incorporated in their entireties
for all purposes). The uptake of such OxLDL by macrophages results
in foam cell generation, which is considered to be an initial-step
in atherosclerosis.
[0005] Endothelial cells under oxidative stress (e.g., in acute
myocardial infarction patients) and activated blood platelets also
produce aldehydes, which interact with the lysine moieties in apo
B-100, resulting in the generation of aldehyde-modified LDL that is
also recognized by the scavenger receptors. However, the lipids in
this aldehyde-modified LDL are not oxidized. Enzymatic activity in
macrophages (e.g. myeloperoxidase) results in the oxidation of both
the lipid and the protein moieties of LDL. All these pathways
result in aldehyde-type modification of the protein moiety of
LDL.
[0006] In vitro experiments and experiments in animal models have
suggested that OxLDL and/or aldehyde-modified LDL may contribute to
the progression of atherosclerosis by inducing endothelial
dysfunction, foam cell generation, smooth muscle cell
proliferation, and platelet activation (for review see document
number 2). A positive correlation between the levels of autoimmune
antibodies that cross-react with aldehyde-modified LDL and the
progression of carotid atherosclerotic lesions in patients
suggested that OxLDL and/or aldehyde-modified LDL might contribute
to the progression of human atherosclerosis (see document 3).
[0007] However, the possibility that the autoimmune antibodies were
directed against other aldehyde-modified proteins, e.g., albumin,
could not be excluded. Therefore, the contribution of OxLDL and
aldehyde-modified LDL (whether or not resulting from oxidation of
the lipid moiety) to human atherosclerosis may be able to be
established when non-invasive tests that are specific for these
substances (i.e., have high affinity for those substances in
preference to other substances) become available.
[0008] Because the underlying mechanisms of oxidation of LDL may be
different in different patient populations (e.g., in diabetes
patients, chronic renal failure patients, heart transplant
patients) and because at least some of the mechanisms may be
independent of lipid oxidation, such tests should be specific for
both OxLDL and aldehyde-modified LDL (e.g., MDA-modified LDL) and
thus preferentially be based on the detection of conformational
changes that specifically occur in the apo B-100 moiety of LDL
following aldehyde-type substitution of lysine residues. In other
words, there is a need for such non-invasive tests (i.e., assays)
that are highly specific for the analytes of interest (i.e.,
MDA-modified LDL and OxLDL). There is also a need for antibodies
that are specific for the analytes of interest. There is also a
need for a stable standard (e.g., to be used as calibrator and/or
control) for the assays.
SUMMARY OF THE INVENTION
[0009] An invention satisfying those needs and having other
features and advantages that will be apparent to those skilled in
the art has now been developed. The present invention provides
antibody-based assays that are capable of specifically quantitating
(quantifying) both OxLDL and aldehyde-modified LDL or MDA-modified
LDL in samples, e.g., samples derived from body fluids (like plasma
or serum) or tissues. The present invention also provides
monoclonal antibodies useful in those assays and cell lines
(hybridomas) that produce those antibodies. The present invention
also provides a storage-stable standard, which can be used as a
calibrator and as a control for the assays. Having such a standard
is necessary for having reliable and reproducible and therefore
useful assays.
[0010] Broadly, in one aspect the present invention concerns an
immunological assay for the detection and/or quantification of
MDA-modified LDL and OxLDL in a sample, said assay comprising:
[0011] a) contacting the sample with a first antibody that has high
affinity for MDA-modified LDL and OxLDL; and
[0012] b) thereafter visualizing and/or quantifying a binding
reaction between the first antibody and the MDA-modified LDL and
OxLDL present in the sample;
[0013] wherein the MDA-modified LDL and OxLDL for which the first
antibody has high affinity contain at least 60 substituted lysine
moieties per apo B-100 moiety.
[0014] That assay may, for example, be a competitive assay, a
sandwich assay, an immunohistochemical assay, etc. "Competitive
assays" are well-known and any competitive assay may be used in
this invention provided it is within the limitations of the
invention and that the benefits of the invention can be achieved.
"Sandwich assays" are well-known and any sandwich assay may be used
in this invention provided it is within the limitations of the
invention and that the benefits of the invention can be achieved.
"Immunohistochemical assays" are well-known and any
immunohistochemical assay may be used in this invention provided it
is within the limitations of the invention and that the benefits of
the invention can be achieved.
[0015] In another aspect, the present invention concerns an
immunological sandwich assay for the detection and/or
quantification of MDA-modified LDL in a sample in which assay a
first antibody that has a high affinity for MDA-modified LDL is
bound to a substrate, said assay comprising:
[0016] (a) contacting the sample with the substrate having bound to
it the first antibody under binding conditions so that at least
some of any MDA-modified LDL in the sample will bind to the first
antibody;
[0017] (b) thereafter removing unbound sample from the
substrate;
[0018] (c) thereafter contacting the substrate with a second
antibody that has a high affinity for MDA-modified LDL; and
[0019] (d) thereafter visualizing and/or quantifying the
MDA-modified LDL that was present in the sample;
[0020] wherein the MDA-modified LDL for which the first antibody
and the second antibody have high affinity contains at least 60
substituted lysine moieties per apo B-100 moiety.
[0021] As used herein (including the claims), "high affinity" means
an affinity constant (association constant) of at least about
5.times.10.sup.8 M.sup.-1, desirably at least about
1.times.10.sup.9 M.sup.-1, preferably at least about
1.times.10.sup.10M.sup.-1, and most preferably of at least about
1.times.10.sup.11 M.sup.-1. As used herein (including the claims),
"low affinity" means an affinity constant (association constant) of
less than about 1.times.10.sup.7 M.sup.-1, desirably less than
about 1.times.10.sup.6 M.sup.-1, and preferably less than about
1.times.10.sup.5 M.sup.-1. Affinity constants are determined in
accordance with the appropriate method described in Holvoet et al.
(4).
[0022] The antibodies that can be used in this invention will bind
with MDA-modified LDL and/or OxLDL whose apo B-100 moieties contain
at least 60, desirably at least about 90, more desirably at least
about 120, preferably at least about 180, more preferably at least
about 210, and most preferably at least about 240 substituted
lysine residues per apo B-100 moiety. The range of lysine
substitution will generally be from 60 to about 240 and preferably
from about 120 to about 240 substituted lysine moieties per apo
B-100 moiety.
[0023] Each new monoclonal antibody is highly specific for a
conformational epitope that is present when at least about 60,
preferably at least about 120 lysine residues, are substituted and
by virtue thereof can distinguish various markers or indications
related to atherosclerosis. Antibodies recognizing epitopes present
when less than about 60 lysines are substituted or modified are
less specific but are still useful (e.g., they may be used as the
secondary antibody in a sandwich ELISA).
[0024] The preferred antibodies used herein are monoclonal
antibodies mAb-4E6, mab-1h11, and mAb-8A2. Their affinity constants
for native LDL, MDA-modified LDL, and OxLDL are as follows:
TABLE-US-00001 Antibody Native LDL MDA-modified LDL OxLDL mAb-4E6
less than 3 .times. 10.sup.10 2 .times. 10.sup.10 1 .times.
10.sup.6 mAb-1H11 less than 3 .times. 10.sup.10 less than 1 .times.
10.sup.6 1 .times. 10.sup.6 mAb-8A2 5 .times. 10.sup.9 1 .times.
10.sup.10 1 .times. 10.sup.10
[0025] In yet another aspect, the present invention concerns (a)
monoclonal antibody mAb-4E6 produced by hybridoma Hyb4E6 deposited
at the BCCM under deposit accession number LMBP 1660 CB on or about
Apr. 24, 1997, (b) monoclonal antibody mAb-8A2 produced by
hybridoma Hyb8A2 deposited at the BCCM under deposit accession
number LMBP 1661 CB on or about Apr. 24, 1997, (c) hybridoma Hyb4E6
deposited at the BCCM under deposit accession number LMBP 1660 CB
on or about Apr. 24, 1997, and (d) hybridoma Hyb8A2 deposited at
the BCCM under deposit accession number LMBP 1661 CB on or about
Apr. 24, 1997.
[0026] The antibodies used in the assays of this invention are
preferably those two (i.e., mAb-4E6 and mAb-8A2) as well as
mAb-1H11. The cell line for antibody mAb-1H11 is produced by
hybridoma Hyb1H11, which was deposited at the BCCM under deposit
accession number LMBP 1659 CB on or about Apr. 24, 1997.
[0027] The BCCM is the Belgian Coordinated Collections of
Microorganisms authorized by the "Budapest Treaty of 28 Apr. 1977
on the International Recognition of the Deposit of Microorganisms
for the Purposes of Patent Procedure." Its address is c/o The
University of Gent, K. Ledeganckstraat 35, B-9000 Gent,
Belgium.
[0028] The assay may be of a type that is well-known, such as an
Enzyme-Linked Immunosorbent Assay (ELISA). For example, in the case
of a sandwich ELISA, mAb-4E6 (for MDA-modified LDL and OxLDL) or
mAb-1H11 (for MDA-modified LDL) may be bound to a solid substrate
and subsequently contacted with a sample to be assayed. After
removal of the sample, binding between the specific antibody and
OxLDL and/or MDA-modified LDL captured out of the sample can be
visualized and/or quantified by detection means. Detection means
may be a labeled, less specific secondary antibody that recognizes
a different part of the apo B-100 moiety of the captured analyte
(e.g., mAb-8A2).
[0029] In the case of a competitive ELISA, a solid substrate coated
with OxLDL or MDA-modified LDL may be contacted for a predetermined
period of time with the monoclonal antibody mAb-4E6 and a sample
thought or known to contain OxLDL and/or MDA-modified LDL, after
which period of time unbound antibody and sample are removed and a
binding reaction between antibody and OxLDL and/or MDA-modified LDL
bound to the substrate is visualized and/or quantified.
Quantification in a competitive ELISA is indirect because the
binding between the antibody and the analyte in the sample is not
measured but instead the amount of antibody that binds to the known
amount of OxLDL or MDA-modified LDL that is coated on (bound to)
the substrate is measured. The more antibody bound to the known
amount of OxLDL or MDA-modified LDL coated on the substrate, the
less analyte there was in the sample.
[0030] In yet another aspect, the present invention concerns a
stable standard containing MDA-modified LDL whose extent of
substitution of its lysine moieties will remain essentially
constant over normal periods of time during normal storage for
biological materials, the MDA-modified LDL of said standard being
made by contacting (incubating) malondialdehyde with LDL at a
predetermined molar ratio of malondialdehyde to the apo B-100
moiety of the LDL.
[0031] "Over normal periods of time during normal storage for
biological materials" as used herein refers to the time periods and
conditions under which biological materials to be used in assays
and other laboratory work are typically stored. Those conditions
will typically include low temperature and in appropriate cases
freezing, either with or without lyophilization. Depending on the
particular biological material, if the material is stored under the
appropriate temperature and other conditions (e.g., lack of
vibration or other movement, proper humidity), the material may be
stable for at least three months, desirably for over a year,
preferably for over two years, and most preferably for over three
years.
[0032] The standard preferably contains an agent that reduces the
ability of any metal ions present to catalyze oxidation of the LDL
(e.g., a chelating agent, such as EDTA) and/or one or more
anti-oxidants (e.g., BHT and/or Vitamin E). Preferably both the
agent that reduces the ability of any metal ions present to
catalyze oxidation of the LDL and the anti-oxidant are used. It has
surprisingly been found that when using an antibody that is
specific for both OxLDL and MDA-modified LDL, the storage-stable
standard of this invention (containing MDA-modified LDL and not
OxLDL) can be used. That eliminates the need to try to formulate,
store, and use a stable standard containing OxLDL. OxLDL may
continue to oxidize under typical storage conditions, making using
as a standard a composition containing OxLDL difficult if not
almost impossible. EDTA will typically be used in concentrations of
0.5 to 5 mM, preferably in concentrations of 0.5 to 2 mM. BHT will
typically be used in concentrations of 5 to 50 .mu.M, preferably in
concentrations of 10 to 20 .mu.M. Vitamin E will typically be used
in concentrations of 5 to 50 .mu.M, preferably in concentrations of
10 to 20 .mu.M. The standard may also contain anti-platelet agents
and coagulation inhibitors.
[0033] It has been found that LDL that has been modified by
treatment with MDA is highly stable. Such MDA-modified LDL (which
is not oxidized, i.e., its lipid moiety is not oxidized) could be
added to reference plasma samples and those samples could be frozen
and thawed without increasing the extent of lysine substitution.
Because the total number of lysine residues in all apo B-100
molecules is identical, a constant MDA/apo B-100 molar ratio in the
reaction mixture will result in an identical number of substituted
lysines in the MDA-modified LDL. In contrast, for example,
metal-ion mediated oxidation of LDL ultimately results in a
variable extent of lysine substitution because it depends on the
oxidation sensitivity of the LDL preparation, which by itself
depends on fatty acid composition and antioxidant content, which
are highly variable even in healthy control individuals.
[0034] As described below, a correlation between the oxidation of
LDL and the extent of post-transplant atherosclerosis in heart
transplant patients was established using this invention. The
relationship between endothelial injury and the modification of LDL
was established in chronic renal failure patients that are at high
risk for atherosclerotic cardiovascular disease. It was also
demonstrated that endothelial injury is an initial step in
atherosclerosis.
[0035] Based on the characteristics of the oxidatively modified LDL
from the plasma of heart transplant and chronic renal failure
patients, it was concluded that cell-mediated aldehyde modification
independent of lipid oxidation was at least partially involved.
This finding further supported the hypothesis that an assay for
oxidatively modified LDL has to detect both OxLDL and
aldehyde-modified LDL.
[0036] In yet another aspect, the invention concerns a kit for
conducting a sandwich assay for the determination of OxLDL or
MDA-modified LDL or both in a sample, said kit comprising a
substrate on which is bound a first antibody that has high affinity
for OxLDL or MDA-modified LDL or both, the OxLDL and MDA-modified
LDL each having at least 60 substituted lysine moieties per apo
B-100 moiety, and a labeled antibody having a high affinity for
OxLDL that becomes bound to the first antibody during the assay or
for MDA-modified LDL that becomes bound to the first antibody
during the assay or for both that become bound to the first
antibody during the assay. Preferably the kit further comprises a
reactive substance for reaction with the labeled antibody (e.g., an
enzyme) to give an indication of the presence of the labeled
antibody. Preferably the kit also comprises the stable standards,
e.g., in the form of stable calibrators and/or stable controls.
Thus, e.g., the bound antibody may be mAb-4E6 or mAb-1H11 and the
labeled antibody may be mab-8A2.
BRIEF DESCRIPTION OF THE DRAWINGS
[0037] The accompanying drawings are provided to help further
describe the invention, which drawings are as follows:
[0038] FIG. 1 illustrates the correlation between amounts of
oxidized and MDA-modified LDL in coronary lesions in Watanabe
heritable hyperlipidemic rabbits (A) and in miniature pigs (B).
[0039] FIG. 2 illustrates the accumulation of OxLDL and
MDA-modified LDL in coronary arteries of cardiac explants of
ischemic heart disease but not of dilated cardiomyopathy
patients.
[0040] FIG. 3 illustrates the inhibition of the binding of mAb-4E6
to immobilized OxLDL by native LDL, OxLDL and MDA-modified LDL in
solution.
[0041] FIG. 4 illustrates a typical standard curve obtained with
MDA-modified LDL in sandwich ELISA.
[0042] FIG. 5 illustrates levels of OxLDL and aldehyde-modified LDL
in posttransplant plasma samples of heart transplant patients with
different extents of angiographically assessed coronary artery
stenosis.
[0043] FIG. 6 illustrates the correlation between plasma levels of
OxLDL and aldehyde-modified LDL and titers of specific
autoantibodies.
[0044] FIG. 7 illustrates the correlation between plasma levels of
OxLDL and aldehyde-modified LDL and of von Willebrand factor
antigen.
[0045] These drawings are provided for illustrative purposes only
and should not be used to unduly limit the invention.
DETAILED DESCRIPTION OF THE INVENTION
[0046] The present invention will be further described in
conjunction with the following examples, which are for illustrative
purposes and which should not be used to unduly limit the
invention.
EXAMPLES
Example 1
Preparation and Characterization of Antibodies Specific for OxLDL
and for Aldehyde-Modified LDL
[0047] Balb/c mice were immunized by intravenous and
intraperitoneal injection of either OxLDL or MDA-modified LDL.
OxLDL was obtained by in vitro incubation of LDL (final apo B-100
concentration 700 .mu.g/ml) with copper chloride (final
concentration 640 .mu.M) for 16 h at 37.degree. C. MDA-modified LDL
was prepared by incubation of LDL (final apo B-100 concentration
700 .mu.g/ml) with a 0.25 M MDA-solution for 3 h at 37.degree. C.
The numbers of substituted lysines, measured in the TBARS assay,
was typically 210 per apo B-100 molecule for OxLDL and 240 for
MDA-modified LDL. Hybridomas were obtained by PEG induced fusion of
spleen lymphocytes derived from immunized mice with P3-X63/Ag-6.5.3
myeloma cells according to standard techniques (4). The screening
for hybridomas producing specific antibodies was performed with
ELISA using microtiter plates coated with malondialdehyde-modified
LDL or copper-oxidized LDL. 308 hybridomas were obtained after
immunization of mice with either OxLDL (211) or MDA-modified LDL
(97). Hyb4E6 produced antibodies specific for both
malondialdehyde-modified and copper-oxidized LDL (mAb-4E6), and
Hyb1H11 produced antibodies specific for malondialdehyde-modified
LDL (mAb-1H11) alone. The IgG fraction of the antibodies was
purified by affinity chromatography on protein A-Sepharose and the
affinity of the purified IgGs was determined in a solid phase
radioimmunoassay and/or in ELISA. The K.sub.a values of the
monoclonal antibody mAb-4E6 were <10.sup.6 M.sup.-1 for native
LDL and >10.sup.9 M.sup.-1 for malondialdehyde-modified LDL and
copper-oxidized LDL. The K.sub.a values for the monoclonal antibody
mAb-1H11 were <10.sup.6 M.sup.-1 for both LDL and OxLDL and
>10.sup.9 M.sup.-1 for malondialdehyde-modified LDL. The K.sub.a
values for the monoclonal antibody mAb-8A2, obtained after
immunization of mice with LDL, were >10.sup.9 M.sup.-1 for all
LDL forms. Delipidation of MDA-modified LDL and OxLDL resulted in a
loss of the immunoreactivity of mAb-4E6, suggesting that it is
directed against a conformational epitope in the protein moiety of
oxidatively modified LDL.
Example 2
Use of mAb-4E6 for the Quantitation of OxLDL and MDA-Modified LDL
in Coronary Lesions of Watanabe Heritable Hyperlipidemic Rabbits
and Miniature Pigs on a Cholesterol Rich Diet
[0048] Coronary arteries were obtained from 2 and 5 month old
Watanabe heritable hyperlipidemic rabbits (n=30) on-normal chow or
from miniature pigs (n=26) which were fed a diet enriched in
cholesterol (4%), saturated fat (14% beef tallow) and bile extract
(1%) for 6 to 24 weeks.
[0049] Arterial specimens were submerged within 30 min after
removal in PBS (pH 7.4) containing 4% sucrose, 20 .mu.M vitamin E
and 10 .mu.M butylated hydroxytoluene as antioxidants, and 1 mM
EDTA, snap-frozen in liquid nitrogen and stored at -80.degree. C.
Frozen 7 .mu.M sections were stained with hematoxylin and eosin and
with oil red 0 or immunostained as described below. Morphometric
parameters of atherosclerotic lesions were measured by planimetry
using the Leica 2 Quantimet color image analyzer (Cambridge, UK).
The area within the external elastic lamina, the internal clastic
lamina and the lumen were measured. Media was defined as the area
between the internal and external clastic lamina. Intima was
defined as the area within the internal elastic lamina not occupied
by vessel lumen.
[0050] Oxidized apo B-100 containing lipoproteins were detected
with the specific monoclonal antibody mAb-4E6, alkaline-phosphatase
conjugated rabbit-anti-mouse IgG antibodies and the fuchsin
alkaline phosphate substrate system (Dako, Carpinteria, Calif.),
and the absorbance was measured in the color image analyzer.
Specificity of immunostaining was confirmed by inhibition of
staining with excess of copper-oxidized LDL but not with native LDL
or with malondialdehyde-modified albumin. The staining co-localized
with that monoclonal antibody mAb-13F6, specific for apo B-100.
Absorbance (approximately 10%) measured with excess copper-oxidized
LDL was presumed to represent background staining.
[0051] FIG. 1 illustrates the correlation between the levels of
oxidized apo B-100 containing lipoproteins, i.e. OxLDL and
MDA-modified LDL, in the lesions and the mean intimal area of
coronary lesions in Watanabe hyperlipidemic rabbits (A) and in
miniature pigs (B). Those data thus demonstrate a correlation
between the accumulation of OxLDL and MDA-modified LDL and the
progression of coronary atherosclerotic lesions in 2 different
animal models. In Watanabe rabbits the progression of the lesions
is due to the increase of LDL cholesterol associated with the
heritable LDL receptor deficiency, whereas the progression in
miniature pigs is due to a diet-induced increase in LDL
cholesterol.
Example 3
Immunohistochemistry
1. Introduction
[0052] This example is a typical example of the use of the highly
specific antibody mAb-4E6 in immunohisto-chemistry applied to human
atherosclerotic lesions. In a similar manner corresponding
experiments may be performed, for which certain conditions can be
adapted by the skilled person using his common knowledge in the
field.
2. Material and Methods
[0053] Coronary artery specimens, obtained at the time of
transplantation from patients with ischemic heart disease (n=7) or
dilated cardiomyopathy (n=7), were treated as described earlier
(document 7). The specimens were collected within 30 min after
removal of the heart in PBS (pH 7.4) containing 4% sucrose, 20
.mu.M vitamin E and 10 .mu.M butylated hydroxytoluene as
antioxidants, and 1 mM EDTA, and were stored at -80.degree. C.
Frozen 7 .mu.m thick sections were cut and stained with hematoxylin
and eosin. Six to 8 sections at a distance of 84 .mu.m were
analyzed for each specimen to insure representative results.
Duplicate slides were developed with monoclonal antibodies mAb-4E6,
specific for oxidized LDL, PG-M1, specific for human macrophages,
or 1A4, specific for human smooth muscle .alpha.-actin (both from
Dako S A, Glostrup, Denmark). Specificity of binding of mAb-4E6 was
confirmed by its inhibition with OxLDL but not with native LDL.
3. Results
[0054] Coronary artery segments of 7 individuals with pretransplant
dilated cardiomyopathy did not contain atherosclerotic lesions and
the monoclonal antibody did not detect OxLDL and/or
aldehyde-modified LDL in these segments. Coronary artery segments
of 7 patients with pretransplant ischemic heart disease all
contained atherosclerotic lesions which contained OxLDL and/or
aldehyde-modified LDL (FIG. 2). This information is sufficient to
state that the antibody detects OxLDL in atherosclerotic lesions in
a highly specific manner.
[0055] OxLDL was associated with macrophage foam cells
(preferentially in lesions with <50% stenosis), with smooth
muscle foam cells and with the necrotic lipid core (preferentially
in lesions with >50% stenosis). Macrophages and smooth muscle
cells were identified by immunostaining with specific monoclonal
antibodies (5). These data supported the hypothesis that oxidation
of LDL may be associated with the development of ischemic coronary
artery disease. The monoclonal antibody mAb-4E6 of the present
invention that detected the immunoreactive material in the tissue
sections was then further used in ELISA (cf. Example 4).
4. Legend to FIG. 2
[0056] Light micrographs (a, c, e; x 40) and phase contrast
micrographs (b, d, f; x 400) of representative left anterior
descending coronary artery specimens of a patient with dilated
cardiomyopathy (male; 40 years of age) (a, b) and of a patient with
ischemic heart disease (male; 57 years of age) (c-f). Tissue
sections were immunostained with the monoclonal antibody mAb-4E6.
Oxidized LDL was undetectable in the neointima of the first patient
(a, b), but demonstrable in plaques of the second patient. The
oxidized LDL was associated with macrophage foam cells that
infiltrated at the shoulder areas of fibrous plaques (c, d) and
with smooth muscle foam cells in fibrous caps (e, f).
Example 4
Competitive ELISA
1. Introduction
[0057] According to the invention an ELISA was established for the
quantitation of OxLDL and aldehyde-modified LDL in plasma. It was
based on the inhibition of the binding of mAb-4E6 to the wells of
microtiter plates coated with copper-oxidized LDL. This antibody
was obtained as described in Example 1.
2. Material and Methods
[0058] Standard OxLDL and aldehyde-modified LDL and plasma samples
were diluted in PBS containing 1 mM EDTA, 20 .mu.M vitamin E, 10
.mu.M butylated hydroxytoluene, 20 .mu.M dipyridamole and 15 mM
theophylline to prevent in vitro LDL oxidation and platelet
activation. Equal volumes of diluted purified mAb-4E6 solution
(final concentration 7.5 ng/ml) and of diluted standard solution
(copper-oxidized LDL added as competing ligand at a final
concentration ranging from 50 to 500 ng/ml) were mixed and
incubated for 30 min at room temperature. Then 200 .mu.l aliquots
of the mixtures were added to wells coated with MDA-modified LDL or
OxLDL.
[0059] Samples were incubated for 2 h at room temperature. After
washing, the wells were incubated for 1 h with horse-radish
peroxidase conjugated rabbit IgG raised against mouse
immunoglobulins and washed again. The peroxidase reaction was
performed as described earlier (5) and the absorbance (A) was read
at 492 nm.
[0060] Controls without competing ligand and blanks without
antibody were included routinely. The percent inhibition of binding
of mAb-4E6 to the immobilized ligand was calculated as: A 492
.times. nm .times. control - A 492 .times. nm .times. sample A 492
.times. control - A 492 .times. blank ##EQU1## and standard curves
were obtained by plotting the percentage of inhibition vs the
concentration of competing ligand.
[0061] The lower limit of detection was 0.020 mg/dl in undiluted
human plasma. Intra- and interassay coefficients of variation were
10 and 12t, respectively. Standard OxLDL and aldehyde-modified LDL
and plasma samples were diluted in PBS containing antioxidants and
antiplatelet agents as described above.
3. Results
[0062] The specificity of mAb-4E6 for OxLDL and aldehyde-modified
LDL is illustrated in FIG. 3. 50% inhibition of binding of mAb-4E6
to immobilized OxLDL and aldehyde-modified LDL was obtained with
0.025 mg/dl copper-oxidized LDL and 25 mg/dl native LDL,
respectively. The C.sub.50 value, i.e., the concentration that is
required to obtain 50% inhibition of antibody binding, increased
from 2.5 mg/dl for MDA-modified LDL with 60 substituted lysine
residues per apo B-100 molecule to 0.025 mg/dl for MDA-modified LDL
with 240 substituted lysine residues per apo B-100 molecule (FIG.
3). Copper-oxidation resulted in fragmentation of the apo B-100
moiety but did not abolish the binding of mAb-4E6 (FIG. 3). 50-fold
higher molar concentrations of MDA-modified albumin were required
to obtain 50% inhibition (not shown), whereas up to 1,000-fold
higher molar concentrations of MDA-modified lysine did not affect
mAb-4E6 binding. OxLDL and aldehyde-modified LDL isolated from
patient plasma had the same reactivity as DA-modified LDL with 120
substituted lysines and as copper-oxidized LDL with 210 substituted
lysines. Intra- and interassay coefficients of variation were 10
and 12%, respectively. When copper-oxidized LDL were added to human
plasma at a final concentration of 0.25 and 2 mg/dl, respectively,
recoveries were 95 and 105%, respectively.
4. Legend to FIG. 3
[0063] Interaction of mAb-4E6 with competing ligands in solution.
Copper-oxidized LDL (1 .mu.g/ml) was the plated antigen. mAb-4E6
was added in the absence and in the presence of competing ligands:
copper-oxidized LDL (V), MDA-modified LDL with 240 (.box-solid.),
120 (.diamond.), 90 (.largecircle.) and 60 (.circle-solid.) blocked
or substituted or modified lysines per apo B-100, respectively,
native LDL (.tangle-solidup.), and OxLDL and aldehyde-modified LDL
(.diamond-solid.) isolated from the plasma of severe chronic renal
failure patients. Results are expressed as B/B.sub.0 where B.sub.0
is the amount of mAb-4E6 bound in the absence and B that amount
bound in the presence of competing ligand.
Example 5
Sandwich ELISA
1. Introduction
[0064] According to the invention a sandwich-type ELISA was
established for the quantitation of OxLDL and aldehyde-modified LDL
in plasma. It was based on the binding of immunoreactive material
to the wells of microtiter plates coated with the monoclonal
antibody mAb-4E6 and the detection of bound immunoreactive material
with the use of the monoclonal antibody mAb-8A2 labeled with
peroxidase. This version of the ELISA is more suited for use in the
clinical laboratory because it overcomes the need to prepare
standard solutions of in vitro oxidized and/or aldehyde-modified
LDL which can only be kept at -4.degree. C. for a limited period of
time, typically 2 weeks. MDA-modified LDL may be added to reference
plasma and those standard preparations may be stored at -80.degree.
C. for up to 1 year (see above).
2. Material and Methods
[0065] Standard preparations and plasma samples were diluted in PBS
containing antioxidants and antiplatelet agents as described above,
180 A1 aliquots of 80-fold diluted plasma and of standard solutions
containing between 10 and 0.01 nM of m alondialdehyde-modified LDL
were applied to the wells of microtiter plates coated with mAb-4E6
(200 .mu.l of a 4 .mu.g/ml IgG solution) and incubated for 2 h at
room temperature. After washing, the wells were incubated for 1 h
with horseradish peroxidase conjugated TAb-8A2, IgG (final IgG
concentration 65 ng/ml) and washed again. The peroxidase reaction
was performed as described above. The absorbance measured at 492 nm
correlates with the log-value of the aldehyde-modified LDL
concentration in the range between 1.5 nM and 0.3 nM.
3. Legend to FIG. 4
[0066] Standard curves for the sandwich ELISA mAb-4E6 was the
plated antibody. MDA-modified LDL was the ligand. Bound
MDA-modified LDL was detected with mAb-8A2 conjugated to horse
radish peroxidase. MDA-modified LDL was added to 8 different plasma
samples to a final concentration of 100 nM and further diluted in
buffer to final concentrations ranging from 2 to 0.2 nM.
Example 6
Use of the ELISA in Diagnosis of Posttransplant Coronary Artery
Disease
1. Introduction
[0067] The ELISA of the present invention was used to study the
association between plasma levels of OxLDL and aldehyde-modified
LDL and posttransplant coronary artery disease.
2. Material and Methods
2.1. Patients
[0068] The posttransplant study group contained 47 patients
transplanted for dilated cardiomyopathy and 60 patients treated for
ischemic heart disease. The clinical characteristics of these
patients are summarized in Table 1. At the time of blood sampling,
between 12 and 84 months after surgery, all patients were in a
stable cardiac condition without evidence of acute rejection. From
14 patients (7 dilated cardiomyopathy and 7 ischemic heart disease
patients) coronary arteries of cardiac explants were isolated and
studied by immunohisto-chemistry (as demonstrated in Example 3).
Adequate information about smoking habits was available for 92 of
the 107 patients (16 smokers and 76 non-smokers). There was no
adequate information about smoking habits of donors. Blood samples
of 53 non-smoking controls (25 males/28 females; age: 52.+-.1.3
years) without a history of atherosclerotic cardiovascular disease
were obtained. The controls were matched for age, gender and levels
of LDL cholesterol. They were selected from the laboratory and
clinical staff.
2.2. Coronary Angiography
[0069] Routine annual coronary angiograms were available for all
posttransplant patients at the time of blood sampling. Coronary
artery disease was independently assessed by two angiographers who
where unaware of the OxLDL and aldehyde-modified LDL levels and was
visually graded as follows: [0070] grade 0: normal coronary
arteries [0071] grade I: minor abnormalities with <50% stenosis
of primary or secondary branches and normal left ventricular
function [0072] grade II: .gtoreq.50% stenosis of primary or
secondary branches, or distal involvement with impaired left
ventricular function. It is well known that angiography
systematically underestimates the extent of coronary intimal
thickening in cardiac transplant recipients. This study therefore
does not attempt to accurately quantify the coronary artery disease
in our patients. Rather the subdivision in groups defined above
relies on angiographic data that are easily distinguishable and
that have been shown to correlate with histopathologic findings.
Out of 107 patients, 46 patients had a normal coronary angiogram 3
years before and development of angiographic, coronary artery
disease within a 3 year follow-up period was assessed in all these
patients. The reference normal coronary angiogram was the first
post-operative angiogram in 18 patients, the second in 14 patients
and the third in 14 patients.
[0073] The study was approved by the Institutional Review Board and
the study subjects provided informed consent.
2.3. Blood Sampling
[0074] Venous blood samples from patients and controls were
collected on 0.1 vol of 0.1 M citrate, containing 1 mM EDTA, 20
.mu.M vitamin E, 10 .mu.M butylated hydroxytoluene, 20 .mu.M
dipyridamole and 15 mM theophylline to prevent in vitro LDL
oxidation and platelet activation. Blood samples were centrifuged
at 3,000 g for 15 min at room temperature within 1 h of collection
and stored at -20.degree. C. until the assays were performed.
2.4. Lipoproteins: Isolation and Modification
[0075] LDL were isolated from pooled sera of fasting normolipidemic
donors by density gradient ultra-centrifugation (document 6).
Standard preparations of MDA-modified and copper-oxidized LDL were
prepared as described elsewhere (7, 8) and were used as assay
controls. Apo B-100 molecules of in vitro MDA-modified LDL (7) and
of copper-oxidized LDL (8) contained on average 244, and 210
substituted lysines (out of a total of 356), respectively (5, 9).
Whereas the extent of lysine-substitution in in vitro MDA-modified
LDL and copper-oxidized LDL is very similar, the lipid moiety of
the former is not oxidized. Specificity of the monoclonal antibody
mAb-4E6 for both MDA-modified LDL and copper-oxidized LDL suggests
that it depends on the extent of protein (lysine) modification
only. All lipoprotein concentrations were therefore expressed in
terms of protein. OxLDL and aldehyde-modified LDL isolated from the
plasma of patients were characterized as described previously (5,
9).
2.5. Assays
[0076] Cholesterol and triglycerides were measured by enzymatic
methods (Boehringer Mannheim, Meylon, France). Typing of major
histocompatibility complex class I (HLA-B) and class II (HLA-DR)
antigen was performed by the microlymphocytotoxicity technique.
[0077] The ELISA of the invention was used to detect OxLDL and
aldehyde-modified LDL.
2.6. Statistical Analysis
[0078] Controls and patients were compared by ANOVA test followed
by nonparameteric Mann-Whitney or Dunnett's multiple comparison
test on logarithmically transformed values, in the Instat V2.05a
statistical program (Graph Pad Software, San Diego, Calif.).
Non-quantitative parameters were compared by Chi-square analysis.
OxLDL and aldehyde-modified LDL levels measured in 3 aliquots of
the same plasma samples were compared in Friedman nonparametric
repeated measures test. Logistic regression analysis, using the SAS
software (SAS Institute Inc., USA), was performed to evaluate the
correlation between angiographically assessed coronary artery
stenosis (as dependent variable) and plasma levels of OxLDL and
aldehyde-modified LDL, age and sex of recipients, age and sex of
donors, pretransplant history of ischemic heart disease or dilated
cardiomyopathy, duration of ischemia, length of follow up, number
of rejections, number of HLA-mismatches, cytomegalovirus infection,
hypertension (antihypertensive treatment), diabetes, treatment with
lipid lowering drugs (statins or fibrates) and serum levels of LDL
cholesterol, HDL cholesterol and triglycerides as independent
variables. p-values of less than 0.05 were considered to indicate
statistical significance. Logistic regression analysis was also
performed to evaluate the correlation between plasma levels of
OxLDL and aldehyde-modified LDL and development of coronary artery
stenosis during a 3-year follow-up period.
3. Results
[0079] The correlation between OxLDL and aldehyde-modified LDL and
coronary artery stenosis was evaluated in 47 patients transplanted
for dilated cardiomyopathy and in 60 patients treated for ischemic
heart disease. Analysis of clinical data for the two groups of
heart transplant patients (Table 1) revealed no significant
differences in age and gender of the recipients, age and gender of
donors, duration of ischemia of the donor heart, number of
rejection episodes, number of HLA-mismatches, frequency of
Cytomegalovirus infections, hypertension or diabetes, and grade of
coronary artery stenosis. Patients transplanted for ischemic heart
disease were followed longer and received more frequently lipid
lowering drugs (Table 1).
[0080] Analysis of the laboratory data (Table 2) revealed no
significant differences in serum levels of triglycerides, HDL
cholesterol and LDL cholesterol between groups of patients or
between patients and controls. However, significant differences in
levels of OxLDL and aldehyde-modified LDL were observed. Mean
plasma levels of OxLDL and aldehyde-modified LDL were 1.3.+-.0.14
mg/dl in dilated cardiomyopathy patients (p<0.001 vs controls)
and 1.7.+-.0.13 mg/dl in ischemic heart disease patients
(p<0.001 vs controls and <0.01 vs dilated cardiomyopathy
patients) (Table 2). Plasma levels of OxLDL and aldehyde-modified
LDL in control subjects matched for age, gender and serum levels of
triglycerides, HDL cholesterol and LDL cholesterol were
0.60.+-.0.034 mg/dl (n=53; p<0.001 vs both transplanted dilated
cardiomyopathy and ischemic heart disease patients).
[0081] Levels of OxLDL and aldehyde-modified LDL were not different
in samples that were stored for 24 h to 4 months after collection,
and up to four thawing and freezing cycles did not cause an
increase of OxLDL and aldehyde-modified LDL levels. These findings
indicated that the addition of EDTA, antioxidants and anti-platelet
agents adequately prevented the in vitro oxidation of LDL. In a
subset of 87 consecutive plasma samples levels of OxLDL and/or
aldehyde-modified LDL were measured in 3 separate aliquots on 3
different days. The levels were 1.30.+-.0.074 mg/dl, 1.48.+-.0.101
mg/dl and 1.46.+-.0.090 mg/dl, respectively. Friedman nonparametric
repeated measures test revealed no significant differences.
[0082] Mean OxLDL and aldehyde-modified LDL levels were
1.2.+-.0.053 mg/dl (n=79) in posttransplant samples of patients
with angiographically normal coronary arteries (grade 0),
2.1.+-.0.30 mg/dl in patients with grade I coronary artery stenosis
(n=18; p<0.001 vs grade 0) and 3.2.+-.0.45 mg/dl in patients
with grade II coronary artery stenosis (n=10; p<0.001 vs grade 0
and p<0.05 vs grade I) (FIG. 5). Serum levels of LDL
cholesterol, triglycerides and HDL cholesterol were very similar in
patients with higher grade of coronary artery stenosis. Levels of
OxLDL and aldehyde-modified LDL in plasma samples of patients
transplanted for dilated cardiomyopathy or ischemic heart disease,
with the same grade of coronary artery stenosis, were similar:
1.1.+-.0.072 and 1.4.+-.0.079 mg/dl for grade 0 patients and
2.6.+-.0.60 and 2.4.+-.0.29 mg/dl for patients with higher grade of
coronary artery stenosis. The number of patients with elevated
levels of OxLDL and aldehyde-modified LDL (>1 mg/dl, i.e. mean
levels of controls+2 SD) were 43 (out of 60) in the subpopulation
of patients with pretransplant ischemic heart disease and 21 (out
of 47) in the subpopulation of patients with pretransplant dilated
cardiomyopathy. Forty-two out of 79 patients with angiographically
normal coronary arteries had elevated levels of OxLDL and
aldehyde-modified LDL. Elevated levels were detected in 12 (out of
18) patients with grade I and in all patients with grade II
stenosis (p=0.0046 for trend).
[0083] To allow further characterization of the immunoreactive
material detected in the ELISA, LDL fractions were isolated from
the plasma of all of 10 patients with grade II coronary artery
stenosis (18). These fractions retained 85.+-.10% (mean.+-.SD) of
the immunoreactive material, whereas no immunoreactive material
migrated in the serum albumin position. OxLDL and aldehyde-modified
LDL were isolated from isolated LDL fractions by ion-exchange
chromatography on a mono Q-Sepharose column with a recovery of 75%.
The number of substituted lysines per apo B-100 molecule was
130.+-.10 for OxLDL and aldehyde-modified LDL compared to 5.+-.1
(p<0.001) for native LDL. The respective cholesterol/protein
ratios were 3.3.+-.0.54 and 1.8.+-.0.36 (p<0.001). The levels of
arachidonate and linoleate in OxLDL and aldehyde-modified LDL
isolated from the plasma of these patients were 75 and 80% lower
than these in native LDL isolated from the same plasma samples. The
inhibition curves obtained with OxLDL and aldehyde-modified LDL
isolated from the plasma of heart transplant patients were
superimposable with these obtained with in vitro oxidized LDL with
the same extent of protein modification (120 substituted lysines
per apo B-100 molecule) (FIG. 3).
[0084] The protein/antigen ratio and the relative reactivity in the
ELISA of OxLDL and aldehyde-modified LDL isolated from the plasma
of these patients were similar to these of copper-oxidized or
MDA-modified standard LDL preparations.
[0085] Logistic regression analysis (Table 3) identified 3
parameters that were significantly and independently correlated
with posttransplant coronary artery stenosis including levels of
OxLDL and aldehyde-modified LDL, length of follow up and donor
age.
[0086] In contrast, pretransplant history of dilated cardiomyopathy
or of ischemic heart disease, age and gender of recipients, gender
of donors, duration of ischemia of the donor heart, extent of
HLA-mismatch, number of rejections, hypertension, diabetes, and
serum levels of LDL cholesterol, HDL cholesterol and triglycerides
in recipients did not significantly contribute to the individual
variations in extent of coronary artery stenosis (Table 3).
[0087] Serum levels of LDL cholesterol, HDL cholesterol and
triglycerides in patients were similar to these in controls (Table
2), so that higher grade of coronary artery stenosis was unlikely
to depend on these variables in this study group. Fifty-six of the
107 transplant patients received lipid lowering drugs (46 with
statins and 10 with fibrates) (Table 1), but the treatment with
these drugs was not correlated with the incidence of angiographic
graft vasculopathy (Table 3). Seventy-five (out of 107) patients
were treated with calcium channel blockers. The plasma levels of
OxLDL and aldehyde-modified LDL in these patients (1.53.+-.0.11
mg/dl) were very similar to these in non-treated patients (1.74
mg/dl) and treatment with these drugs was not correlated with the
extent of coronary artery stenosis.
[0088] Development of coronary artery disease was observed in 12
out of 46 heart transplantation patients during a 3-year follow-up
period. There were no differences in age and gender of recipients,
age and gender of donors, duration of ischemia, extent of HLA
mismatch, frequency of cytomegalovirus infections, hypertension and
diabetes (Table 4) nor in serum levels of triglycerides, HDL
cholesterol and LDL cholesterol (Table 5) between patients without
and with development of coronary artery disease. However, levels of
OxLDL and aldehyde-modified LDL were significantly elevated in
patients with development of coronary artery disease (Table 5).
[0089] Logistic regression analysis revealed that plasma levels of
OxLDL and aldehyde-modified LDL (Chi-square value=7.1; p=0.0076)
and age of donor (Chi-square value=4.4; p=0.035) predicted the
development of coronary artery disease in these patients. Three of
these patients developed coronary artery disease in the first year,
3 in the second and 6 in the third year. The plasma levels of OxLDL
and aldehyde-modified LDL were 3.9.+-.0.6 mg/dl, 2.0.+-.0.37 mg/dl
and 1.2.+-.0.33 mg/dl, respectively. Although statistical analysis
showed no correlation with gender, hypertension and Cytomegalovirus
infection, 8 out of 12 of these patients were male, hypertensive
and had Cytomegalovirus infection.
4. Discussion
[0090] This demonstrates:
[0091] 1) that cardiac explants of patients with ischemic heart
disease, but not with dilated cardiomyopathy, contain oxidized LDL
in macrophages and in smooth muscle cells in atheromatous
plaques;
[0092] 2) that posttransplant coronary artery disease is associated
with increased plasma levels of OxLDL and aldehyde-modified LDL
both in patients transplanted for dilated cardiomyopathy or for
ischemic heart disease, and
[0093] 3) that increased plasma levels of OxLDL and
aldehyde-modified LDL correlate with the development of coronary
artery stenosis.
[0094] OxLDL and aldehyde-modified LDL levels in plasma samples of
heart transplant patients without angiographically detectable
coronary artery lesions were 2-fold higher than in plasma samples
of control subjects without a history of atherosclerotic
cardiovascular disease, who were matched for age, gender, and
plasma levels of LDL cholesterol, HDL cholesterol and
triglycerides. A further 2.7-fold increase was observed in
posttransplant plasma samples of patients with pronounced coronary
artery stenosis. These data suggest that elevated plasma levels of
OxLDL and aldehyde-modified LDL may be an indicator of
posttransplant coronary artery stenosis. Increased plasma levels of
OxLDL and aldehyde-modified LDL correlated with the extent of
coronary artery stenosis and also with its progression, suggesting
that OxLDL and aldehyde-modified LDL may play a pathogenic role in
the accelerated progression of coronary artery disease in heart
transplant patients.
[0095] It has been suggested that posttransplant atherosclerosis
results from a "response to injury" of the endothelium (10). The
extent of ischemic injury in endomyocardial biopsies was indeed
found to be a strong predictor of the development of accelerated
atherosclerosis (11-13). Endothelial injury may be induced by
cellular delayed-type hypersensitivity immune responses elicited by
class II histocompatibility (HLA) antigens on coronary artery
endothelium (14), by cytomegalovirus infection (15, 16), by
cyclosporin (17) and by OxLDL and aldehyde-modified LDL (18) that
may act synergistically with cyclosporin (19). In the present
study, the extent of histoincompatibility between pairs of donors
and recipients, the number of episodes of rejection or
Cytomegalovirus infection did not correlate with the grade of
coronary artery stenosis, whereas OxLDL and aldehyde-modified LDL
were significantly and independently correlated with posttransplant
coronary artery disease. The observed association between the age
of the donor and the occurrence of coronary artery disease is in
agreement with previous findings that coronary atherosclerosis in
the donor heart predisposes to accelerated posttransplant coronary
artery stenosis (20).
[0096] OxLDL and aldehyde-modified LDL were demonstrated in
coronary arteries in cardiac explants of ischemic heart disease
patients suggesting that OxLDL and aldehyde-modified LDL that
accumulate in the arterial wall may contribute to the progression
of coronary artery stenosis. The cholesterol/protein ratio in OxLDL
and aldehyde-modified LDL was very similar to that in LDL extracted
from atherosclerotic lesions as described previously (21,22). A
possible explanation is that at least part of the OxLDL and
aldehyde-modified LDL is released from the arterial wall.
Previously, we have demonstrated that plaque rupture in acute
myocardial infarction patients is associated with the release of
oxidatively modified LDL (5).
[0097] In vitro data suggest that OxLDL and aldehyde-modified LDL
may be linked to atherogenesis by a sequence of events (reviewed in
2,23). Endothelial cells exposed to OxLDL and aldehyde-modified LDL
secrete adhesion molecules, chemoattractant proteins and
colony-stimulating factors that enhance the infiltration,
proliferation and accumulation of monocytes/macrophages in the
arterial wall. Uptake of OxLDL and aldehyde-modified LDL by
infiltrated macrophages may result in the generation of foam cells
that produce oxygen radicals and thus further contribute to the
oxidation of LDL. It has been demonstrated that OxLDL and
aldehyde-modified LDL inhibit the migration of aortic endothelial
cells in vitro, suggesting that OxLDL and aldehyde-modified LDL may
limit the healing response of the endothelium after injury, and
that basic fibroblast growth factor reverses the atherosclerosis
associated impairment of human coronary angiogenesis-like responses
in vitro (24,25). OxLDL and aldehyde-modified LDL may also
contribute to rapidly progressing coronary atherosclerosis by
inducing platelet adhesion, by decreasing the anticoagulant and
fibrinolytic capacities of activated endothelium and by impairing
vasodilation and inducing shear stress (2,23).
[0098] Increased intracellular levels of ferritin (26) or of
alpha-tocopherol analogs (27) decreased the extent of endothelial
injury elicited by OxLDL and aldehyde-modified LDL in vitro,
whereas antioxidants protect against progression of atherosclerosis
in experimental animals (reviewed in document 28).
[0099] In summary, the present example demonstrates that
posttransplant atherosclerosis correlates with plasma levels of
OxLDL and aldehyde-modified LDL.
5. Legend to the FIG. 5
[0100] Plasma levels of OxLDL and aldehyde-modified LDL and
angiographically assessed grade of coronary artery stenosis. Grade
0: normal coronary arteries; grade I: minor abnormalities with
<50% stenosis of primary or secondary branches and normal left
ventricular function; and grade II: .gtoreq.50% stenosis of primary
or secondary branches, or distal occlusions with impaired left
ventricular function.
Example 7
Use of the ELISA in Renal Failure Patients
1. Material and Methods 1.1. Subjects
[0101] The patient population consisted of 20 mild chronic renal
failure (MCRF) and 77 severe chronic renal failure patients: 21 on
conservative treatment including dietary and antihypertensive
treatment (SCRF), and 56 on a four-hour, three times a week
hemodialysis schedule (HEMO) for 66 months (95% CI, 50-82 months).
All hemodialysis patients were given an oral polyvitamin
preparation (01-Amine, La Meuse, Belgium) after hemodialysis, which
contained only minute amounts of antioxidant compounds (i.e. 5 mg
of vitamin E and 100 mg of vitamin C). Controls and non-dialyzed
patients did not receive routine prescriptions of vitamin
supplements. The high frequency of atherosclerotic disease in these
patients (Table 6) is in agreement with previously published data
(29, 30). The diagnosis of atherosclerotic heart disease,
cerebrovascular disease and peripheral vascular disease was made
after reviewing the patient files for a history of myocardial
infarction, unstable angina or antianginal treatment,
cerebrovascular accidents, transient ischemic attack or events
related to peripheral vascular disease such as ischemic ulcera,
amputation or bypass surgery. Angiograms were available for only a
few patients. No patients had evidence of unstable atherosclerotic
disease at the time of blood sampling nor in the following days. A
group of 27 healthy volunteers (Table 6) without a history of renal
disease or atherosclerotic vascular disease served as controls.
Patients receiving lipid lowering drugs were excluded. The study
was approved by the Institutional Review Board and the study
subjects provided informed consent.
1.2. Blood Samples
[0102] Venous blood samples from patients and controls were
collected on 0.1 vol of 0.1 M citrate, containing 1 mM EDTA, 20
.mu.M vitamin E, 10 .mu.M butylated hydroxytoluene, 20 .mu.M
dipyridamole and 15 mM theophylline to prevent in vitro LDL
oxidation and in vitro platelet activation, respectively. Blood
samples were centrifuged at 3,000 g for 15 min at room temperature
within 1 h of collection and stored at -20.degree. C. until the
assays were performed.
1.3. Assays
[0103] Titers of autoantibodies against OxLDL and aldehyde-modified
LDL and native LDL were measured according to Salonen et al. (3) as
described in detail elsewhere (5). vWF antigen levels were measured
in a sandwich-type ELISA based on a polyclonal rabbit anti-human
vWF antiserum (Dako, Glostrup, Denmark), horseradish
peroxidase-conjugated rabbit anti-human vWF IgG (Dako) and
o-phenylenediamine. Plasma levels of total cholesterol, HDL
cholesterol and triglycerides were determined using standard
enzymatic assays (Boehringer Mannheim, Meylon, France). The LDL
cholesterol levels were calculated using the Friedewald formula.
For the patients not in hemodialysis, creatinine clearance rates
were calculated from plasma creatinine levels using the Cockcroft
and Gault formula (31).
11.4. Statistical Analysis
[0104] Controls and patients were compared by ANOVA test followed
by Dunnett's multiple comparison test, in the Instat V2.05a
statistical program (Graph Pad Software, San Diego, Calif.).
Correlation coefficients were calculated according to Spearman.
Multiple regression analysis, using the SAS software (SAS Institute
Inc., USA), was performed to study the relationship between OxLDL
and aldehyde-modified LDL as dependent variable, and age, sex,
hypertension (antihypertensive treatment), levels of triglycerides,
HDL cholesterol, LDL cholesterol and creatinine clearance rates
(marker of extent of renal failure) and levels of vWF (marker of
endothelial injury) as independent variables.
2. Results
[0105] Mean plasma levels of OxLDL and aldehyde-modified LDL in
controls were 0.59 mg/dl (95% CI, 0.52-0.66 mg/dl; n=27), and were
2.7-fold higher in MCRF patients (p<0.01 as determined by
Dunnett's multiple comparison test), 3.1-fold higher in SCRF
patients (p<0.001), and 5.4-fold higher in HEMO patients
(p<0.001) (Table 7). OxLDL and aldehyde-modified LDL levels were
inversely correlated with creatinine clearance rates (r=-0.65;
p<0.001; n=73). HEMO patients were not included in this analysis
because their plasma creatinine clearance cannot be determined
adequately.
[0106] In a series of 14 hemodialyzed patients, levels of OxLDL and
aldehyde-modified LDL were found to be very similar in fresh and in
fresh frozen plasma samples. Three freezing and thawing cycles did
not cause an increase of OxLDL and aldehyde-modified LDL,
indicating that addition of antioxidants and antiplatelet agents
prevented in vitro oxidation.
[0107] Plasma samples were obtained from 14 hemodialyzed patients
on 3 consecutive days before the start of the dialysis procedure.
The levels of OxLDL and aldehyde-modified LDL in these samples were
similar: 3.4.+-.0.25 mg/dl, 3.2.+-.0.21 mg/dl and 3.5.+-.0.28
mg/dl, respectively. Furthermore, plasma samples were obtained
during (after 2 h) and at the end (after 4 h) of hemodialysis.
Plasma levels of OxLDL and aldehyde-modified LDL were 4.0.+-.0.60
mg/dl and 4.7.+-.0.70 mg/dl (p=NS vs before) as compared to
3.4.+-.0.25 mg/dl before the start of the dialysis procedure. Thus
the hemodialysis procedure did not induce a significant increase in
the OxLDL and aldehyde-modified LDL levels.
[0108] Adequate information about smoking habits was only available
for controls (27 non-smokers) and for HEMO patients (12 smokers and
45 non-smokers). Levels of OxLDL and aldehyde-modified LDL were
somewhat higher in smoking HEMO patients (3.6 mg/dl; 95% CI,
2.1-5.6 mg/dl) than in non-smoking HEMO patients (3.0 mg/dl; 95%;
CI, 2.5-3.6 mg/dl; p=NS). The plasma levels of OxLDL and
aldehyde-modified LDL in hemodialyzed patients with a history of
unstable atherosclerotic cardiovascular disease were 3.5.+-.0.40
mg/dl (n=30) as compared to 2.8.+-.0.60 mg/dl (n=26, p=NS) in
hemodialyzed patients without a history of unstable atherosclerotic
cardiovascular disease.
[0109] LDL fractions were isolated from the plasma of 10 controls,
of 10 MCRF patients, of 10 SCRF-patients and of 10 HEMO patients by
gel filtration on a Superose 6HR 10/30 column, as described
previously (5). 75.+-.6% (mean.+-.SD), 80.+-.4%, 83.+-.6% and
79.+-.5% of the immunoreactive material was recovered in the LDL
fractions. No immunoreactive material migrated in the serum albumin
position. The inhibition curves obtained with the respective LDL
fractions were parallel to those obtained with in vitro
copper-oxidized or MDA-modified standard LDL preparations. OxLDL
and aldehyde-modified LDL were isolated from isolated LDL fractions
of 10 SCRF patients by ion-exchange chromatography on a mono
Q-Sepharose column with a recovery of 75%. Their physicochemical
properties are summarized in Table 8. The levels of arachidonate of
OxLDL and aldehyde-modified LDL isolated from these patients were
reduced with 75%, whereas its linoleate levels were reduced with
80%. Thirty-seven % of the lysine residues of OxLDL were
substituted with aldehydes. The inhibition curves obtained with
OxLDL and aldehyde-modified LDL isolated from the plasma of chronic
renal failure patients were parallel to these obtained with OxLDL
and aldehyde-modified LDL that was obtained by in vitro oxidation
of LDL that had been isolated from the plasma of control subjects
(FIG. 3). The protein/antigen ratio and the relative reactivity in
the ELISA of OxLDL and aldehyde-modified LDL isolated from the
plasma of these patients were similar to these of copper-oxidized
or MDA-modified standard LDL preparations (Table 8).
[0110] Titers of autoantibodies against OxLDL and aldehyde-modified
LDL were 4.2 (95% CI, 4.0-4.4) in controls, were similar in MCRF
and SCRF patients, but significantly increased in HEMO patients
(p<0.001) (Table 7). Autoantibody titers correlated with levels
of OxLDL and aldehyde-modified LDL in SCRF patients (r=0.44;
p=0.047) and in HEMO patients (r=0.37; p=0.0055) (FIG. 6). No
circulating autoantibodies against native LDL could be
detected.
[0111] Levels of vWF were 100 percent in controls (95% CI, 90-110
percent), and were 1.5-fold higher in MCRF patients (p=NS vs
controls), 1.6-fold higher in SCRF patients (p<0.01) and
2.1-fold higher (p<0.001) in HEMO patients (Table 7). Levels of
vWF were not significantly higher in smoking HEMO patients (250
percent; 95%, 150-340 percent; n=12) than in non-smoking HEMO
patients (220 percent; 95% CI, 190-260 percent; n=45). Levels of
vWF correlated with levels of OxLDL and aldehyde-modified LDL in
MCRF patients (r=0.59; p<0.0057), in SCRF patients (r=0.69;
p=0.0006) and in HEMO patients (r=0.62; p<0.0001) (FIG. 7). In
contrast, levels of vWF did not correlate with LDL cholesterol
levels or with body weight.
[0112] Multiple regression analysis revealed that the extent of
renal failure (F=14; p=0.0004) and the extent of endothelial injury
(F=26; p=0.0001), but not age, sex, hypertension, triglyceride
levels, HDL cholesterol or LDL cholesterol levels, accounted for a
significant fraction of the variations in OxLDL and
aldehyde-modified LDL levels (Table 9). Even when only subjects
without evidence of ischemic atherosclerotic disease (n=53) were
included in the model (R.sup.2-value=0.68) only the extent of renal
failure (F=21; p=0.0001) and the extent of endothelial injury
(F=14; p=0.0006) contributed significantly to the variations in
OxLDL and aldehyde-modified LDL levels. No other variables
contributed significantly to these variations after exclusion of
subjects without evidence of ischemic atherosclerotic disease. When
only subjects with evidence of ischemic atherosclerotic disease
(n=15) were included only the extent of endothelial injury (F=6.2;
p=0.047; R.sup.2-value=0.65) contributed to the variations in OxLDL
and aldehyde-modified LDL levels. Exclusion of diabetic patients
did not significantly change the data either. After exclusion of
the extent of renal failure as an independent variable, multiple
regression analysis revealed that hemodialysis (F=5.6; p=0.021;
n=77), LDL cholesterol levels (F=7.1; p=0.0095) and endothelial
injury (F=35; p=0.0001) accounted for a significant fraction of the
variation in OxLDL and aldehyde-modified LDL levels in severe
chronic renal failure patients (Table 10).
3. Discussion
[0113] In vitro work and experimental animal data suggest that
oxidized LDL (OxLDL and aldehyde-modified LDL) may contribute to
the progression of atherosclerosis (reviewed in document 2), and
OxLDL and aldehyde-modified LDL have been demonstrated in human
atherosclerotic plaques (5). The immuno-assay of this invention
identifies OxLDL and aldehyde-modified LDL (MDA-modified LDL) with
260 substituted lysines per apo B-100 molecule, which represents
the threshold of substitution required for scavenger receptor
mediated uptake (1). Increased levels of OxLDL and
aldehyde-modified LDL have been measured by ELISA in the plasma of
chronic renal failure patients.
[0114] Overall, 80 percent of the immunoreactive material isolated
from the plasma of patients was recovered in the LDL fractions that
were separated by gel filtration. No immunoreactive material
migrated in the albumin position. Inhibition curves obtained with
the isolated OxLDL and aldehyde-modified LDL were parallel to these
of in vitro copper-oxidized or MDA-modified LDL standard
preparations and the protein/antigen ratio and the C.sub.50 value
of the isolated OxLDL and aldehyde-modified LDL were identical to
these of standard OxLDL and aldehyde-modified LDL preparations.
These data suggested that increased immunoreactivity of OxLDL and
aldehyde-modified LDL fractions in plasma of these patients with
the antibodies of this invention depended indeed on the higher
extent of protein modification and not on changes in lipid
composition as was previously observed with other antibodies (32).
The increased electrophoretic mobility, the increased lysine
modification, the increased cholesterol/protein ratio, the
decreased arachidonic acid and linoleate levels were very similar
to these of modified LDL extracted from atherosclerotic lesions
(21, 22). OxLDL and aldehyde-modified LDL induced foam cell
generation, suggesting that OxLDL and aldehyde-modified LDL were
not "minimally modified" LDL.
[0115] Multiple regression analysis revealed that chronic renal
failure and endothelial injury contributed significantly to the
variation in OxLDL and aldehyde-modified LDL levels even when
patients with evidence of ischemic atherosclerotic disease were
excluded. Indeed, 79.6% and 82.4% of the variations in OxLDL and
aldehyde-modified LDL levels could be explained in these models. No
patients had evidence of unstable atherosclerotic disease at the
time of blood sampling nor in the following days and exclusion of
patients with a history of ischemic atherosclerotic disease did not
affect the contribution of the extent of renal failure and of
endothelial injury to the variations in OxLDL and aldehyde-modified
LDL.
[0116] LDL cholesterol levels in controls and patients were very
similar and LDL cholesterol levels did not contribute to the
variations in OxLDL and aldehyde-modified LDL levels. Sutherland et
al. (33) demonstrated that the lag time of conjugated diene
formation, which is a measure for the sensitivity of LDL to in
vitro oxidation, was similar in patients with chronic renal failure
and in matched controls. The maximum rate and the extent of LDL
oxidation were even lower in patients with renal disease than in
controls, due to lower levels of linoleic acid and higher levels of
oleic acid. Furthermore, Schulz et al. (34) demonstrated that
despite the fact that hemodialysis causes leukocyte activation, the
in vitro LDL oxidation lag time was similar in renal patients and
in healthy controls. It was concluded that the antioxidative
defense of lipoproteins was preserved in renal failure and during
dialysis.
[0117] In experimental models, antioxidants such as probucol and
vitamin E were found to protect against glomeral injury (35, 36)
and to slow atherogenic processes (28). Renal vasoconstriction
caused by cholesterol feeding was corrected by probucol or by a
thromboxane antagonist (35). Galle et al. (38) demonstrated that
the inhibition of endothelium-dependent dilation induced by
oxidized lipoprotein could be prevented by high density
lipoproteins that are significantly decreased in hemodialyzed
patients. In addition, minerals like selenium and nutrients such as
coenzyme Q10 may minimize free radical generation and thus
oxidative stress. Folic acid, vitamin B12 and vitamin B6 may be
essential in the prevention of hyperhomocysteinemia that may
contribute to the endothelial injury (39) and to oxidation of LDL
(40) in these patients. A diet rich in mono-unsaturated fatty acids
(oleic acid, resistant to oxidation) reduced the extent of
endothelial injury in diabetes patients (41). Thus it is possible
that dietary or pharmacological means may reduce OxLDL and
aldehyde-modified LDL and von Willebrand factor in chronic renal
failure and alleviate the enhanced generalized atherosclerosis in
such patients.
[0118] After adjustment for the extent of renal failure, multiple
regression analysis revealed that both LDL cholesterol levels and
endothelial injury strongly contributed to the variations in OxLDL
and aldehyde-modified LDL levels in severe chronic renal failure
patients.
[0119] Hemodialysis results in platelet and leukocyte activation
(42, 43), which generates oxygen radicals and aldehydes that may
also contribute to oxidation of LDL. OxLDL and aldehyde-modified
LDL may then contribute to thrombogenesis and atherogenesis by
stimulating platelets (44). Because of the rather limited number of
patients, subgroup analysis to further study the interaction
between hemodialysis, oxidation of LDL and ischemic atherosclerotic
disease could not be performed (45).
4. Legend to FIGS. 6 and 7
[0120] FIG. 6. Correlation between plasma levels of OxLDL and
aldehyde-modified LDL (log values) and titers of autoantibodies
(log values): regression line for severe chronic renal failure
patients, either on conservative treatment (.tangle-solidup.; ---)
(r=0.44; p=0.047) or on hemodialysis (.box-solid.; -) (r=0.37;
p=0.0055). No significant correlation was observed in controls and
in mild chronic renal failure patients.
[0121] FIG. 7. Correlation between plasma levels of OxLDL and
aldehyde-modified LDL (log values) and of von Willebrand factor
antigen (log values): regression line for mild chronic renal
failure patients (.circle-solid.; -.-.-.) (r=0.59; p=0.0057) or for
severe chronic renal failure patients either on conservative
treatment (.tangle-solidup.; ---) (r=0.69; p=0.0006) or on
hemodialysis (.box-solid.; -) (r=0.62; p<0.00001). No
significant correlation was observed in controls.
Example 8
Preparation of Reference-Standard for Use in Immunological
Assays
1. Introduction
[0122] According to the invention it has been found that LDL that
is modified by treatment with malondialdehyde (MDA) is highly
stable. Furthermore, the extent of modification is highly
reproducible. LDL modified with MDA in a particular ratio has an
identical number of substituted lysines and can therefore be used
as a reference sample in immunological assays. This example shows
the preparation of the standard.
2. Material and Methods
[0123] MDA-modified LDL was added to control plasma (containing
anti-oxidants and anti-platelet compounds and anti-coagulants) to a
final concentration of 10 nM MDA modified apo B-100. Aliquots were
frozen at -80.degree. C. In 6 days were aliquots were thawed,
diluted to final concentrations ranging from 10 to 0.1 mM
MDA-modified apo B-100 and analyzed in ELISA (4 dilution curves per
day).
3. Results
[0124] The inter-assay variation coefficients of 10 subsequent
sandwich ELISA's of this invention using 10 subsequent, independent
MDA-modified LDL standard preparations of this invention are
summarized in Table 11.
[0125] These data show that for concentrations of MDA-modified LDL
ranging from 10 and 0.01 mM the inter-assay variation ranged from
7.6 to 16.9%.
Abbreviations
[0126] C.sub.50 concentration required to obtain 5011 inhibition of
antibody binding [0127] MDA: malondialdehyde [0128] HEMO: severe
chronic renal failure patients on maintenance hemodialysis [0129]
MCRF: mild chronic renal failure patients [0130] SCRF: severe
chronic renal failure patients on conservative treatment
[0131] OxLDL: oxidized low density lipoproteins. TABLE-US-00002
TABLE 1 Clinical data of heart transplant patients Heart transplant
patients Dilated cardiomyopathy Ischemic heart disease
Characteristics (n = 47) (n = 60) p-values Age of recipient (yr) 54
.+-. 1.6 55 .+-. 0.95 *NS Gender of recipient (M/F) 41/6 53/7 *NS
Age of donor (yr) 29 .+-. 1.5 29 .+-. 1.4 **NS Gender of donor
(M/F) 31/16 44/16 *NS Length of follow up (mo) 39 .+-. 3.1 50 .+-.
2.7 **0.008 Duration of ischemia (min) 130 .+-. 7.0 140 .+-. 5.3
**NS No of HLA mismatches DR 1.5 .+-. 0.09 1.4 .+-. 0.08 **NS B +
DR 3.1 .+-. 0.13 3.0 .+-. 0.13 **NS No of rejection episodes 0.38
.+-. 0.13 0.25 .+-. 0.06 **NS Cytomegalovirus infection 26 43 *NS
Hypertension 37 53 *NS Diabetes 4 3 *NS Coronary artery disease
Grade 0 39 40 *NS Grade I 5 13 *NS Grade II 3 7 *NS Lipid lowering
drugs 17 39 *0.004 Statins 13 33 *0.006 Fibrates 4 6 *NS Calcium
channel blockers 31 47 *NS Data represent mean .+-. SEM or number
of patients. *p-values determined by Chi-square test. **p-values
determined by Dunnett's multiple comparison test. NS: not
significant.
[0132] TABLE-US-00003 TABLE 2 Laboratory data of controls and heart
transplant patients Heart transplant patients Dilated Controls
cardiomyopathy (DC) Ischemic heart disease Characteristics (n = 27)
(n = 47) p vs control (n = 60) p s control p-valuesvs DC Serum
triglyderides (mg/dl).dagger. 130 .+-. 11 130 .+-. 8.3 NS 140 .+-.
7.0 NS NS HDL cholesterol (mg/dl).dagger-dbl. 44 .+-. 2.1 54 .+-.
2.5 NS 49 .+-. 1.9 NS NS LDL cholesterol (mg/dl).dagger-dbl. 120
.+-. 4.7 100 .+-. 4.4 NS 110 .+-. 3.3 NS NS Oxidized LDL (mg/dl)
0.59 .+-. 0.036 1.3 .+-. 0.14 <0.001 1.7 .+-. 0.13 <0.001
<0.01 Data represent mean .+-. SEM. p-values determined by
Dunnett's multiple comparison test. NS: not significant. .dagger.to
convert values for serum triglycerides to millimoles per liter,
multiply by 0.011. .dagger-dbl.to convert values for serum
cholesterol to millimoles per liter, muliply by 0.026.
[0133] TABLE-US-00004 TABLE 3 Logistic regression analysis of the
relation between clinical- laboratory data and extent of coronary
artery stenosis in heart transplant patients. Independent variable
Chi-square value p-value Oxidised LDL 18 0.0001 Length of follow up
11 0.0008 Age of donor 3.9 0.047 Age of recipient 0.12 0.73 Sex of
recipient 1.8 0.18 Sex of donor 0.025 0.88 History of pretransplant
dilated 0.0018 0.97 cardiomyopathy (n = 47) or ischemic heart
disease (n = 60) Duration of ischemia 0.25 0.62 No of HLA
mismatches 1.6 0.20 No of rejection episodes 3.0 0.081
Cytomegalovirus infection 0.17 0.47 Hypertension 1.9 0.16 Diabetes
0.0016 0.97 Treatment with lipid lowering drugs Statins 1.1 0.30
Fibrates 0.12 0.73 Treatment with calcium channel blockers 0.16
0.49 Serum triglycerides 0.18 0.67 Serum HDL cholesterol 0.25 0.61
Serum LDL cholesterol 0.044 0.83 The data set contained 107
patients. Original cardiac disease was dilated cardiomyopathy in 47
and ischemic heart disease in 60 patients. Coronary artery stenosis
was assessed angiographically. All quantitative parameters were
transformed logarithmically to obtain a normal distribution for
linear regression. Chi-square values were obtained after adjustment
for all other variables.
[0134] TABLE-US-00005 TABLE 4 Clinical data of heart transplant
patients without and with progression of coronary artery stenosis
during a 3 years follow-up period. Heart transplant patients With
Without progression progression Characteristics (n = 34) (n = 12)
p-value Age of recipient (yr) 58 .+-. 1.4 60 .+-. 1.4 **NS Gender
of recipient (M/F) 21/14 11/1 *NS Age of donor (yr) 25 .+-. 1.3 32
.+-. 3.8 **NS Gender of donor (M/F) 27/7 10/2 *NS Duration of
ischemia (min) 130 .+-. 6.7 140 .+-. 11 **NS No of HLA mismatches
DR 1.2 .+-. 0.13 1.5 .+-. 0.15 **NS B + DR 2.8 .+-. 0.21 3.2 .+-.
0.24 **NS Cytomegalovirus infection 24 11 *NS Hypertension 21 10
*NS Diabetes 1 2 *NS Data represent mean .+-. SEM or number of
patients. *p-values determined by Chi-square analysis. **p-values
determined by Dunnett's multiple comparison test. NS = not
significant
[0135] TABLE-US-00006 TABLE 5 Laboratory data of heart transplant
patients without and with progression of coronary artery stenosis
during a 3 years follow-up period. Heart transplantation patients
With Without progression progression Characteristics (n = 34) (n =
12) p-value Serum triglycerides (mg/dl) 130 .+-. 8.6 150 .+-. 14 NS
HDL cholesterol (mg/dl) 50 .+-. 2.7 49 .+-. 4.9 NS LDL cholesterol
(mg/dl) 110 .+-. 3.6 105 .+-. 8.7 NS Oxidized LDL (mg/dl) 1.2 .+-.
0.069 2.6 .+-. 0.33 0.0005 Data represent means .+-. SEM. p-values
determined by Dunnett's multiple regression comparison test. NS =
not significant.
[0136] TABLE-US-00007 TABLE 6 Mild chronic Severe chronic renal
failure Controls renal failure non-dialysed hemodialysed
Characteristics (n = 27) (n = 20) (n = 21) (n = 56) Males/females
12/15 11/9 7/14 33/23 Age (years) 54 (50-58)* 52 (44-60)* 55
(49-62)* 61 (58-65)* Body weight (kg) 72 (69-76)* 73 (67-79)* 59
(53-65)* 65 (61-68)* Creatinine clearance 110 (110-120)* 34
(29-39)* 8.4 (7-10)* nd (ml/min) Primary renal disease:
Glomerulonephritis -- 4 3 11 Autosomal dominant polycystic -- 2 6
10 kidney disease Diabetes -- 1 4 6 Reflux-nephropathy -- 1 2 2
Chronic Interstitial Nephritis -- 2 2 9 Hypertensive nephropathy --
2 0 2 Other.sup.1 -- 6 1 9 Unknown -- 2 3 7 Hypertension 1 16 19 18
Atherosclerotic heart disease -- 6 7 24 Cerebrovascular accidents
-- 0 3 9 Peripheral vascular disease -- 2 1 13 *Data represent
means and 95% confidence intervals (between brackets).
.sup.1including: hereditary nephropathy, sarcoidosis, renal
tuberculosis, thrombotic thrombocytopenic purpura, myeloma,
traumatic loss, congenital urinary tract abnormalities. nd:
creatinine clearance rate cannot be determined adequately.
[0137] TABLE-US-00008 TABLE 7 Laboratory data of study subjects
Controls MCRF patients SCRF patients HEMO patients (n = 27) (n =
20) p vs controls (n = 21) p vs controls (n = 56) p vs controls
Triglycerides (mg/dl) 120 (100-150) 150 (130-170) NS 120 (100-140)
NS 130 (110-160) NS HDL cholesterol (mg/dl) 44 (39-48) 38 (33-42)
NS 44 (38-50) NS 37 (35-40) <0.05 LDL cholesterol (mg/dl) 120
(110-130) 110 (100-130) NS 110 (105-130) NS 120 (110-130) NS
Oxidized LDL (mg/dl) 0.59 (0.52-0.66) 1.6 (1.0-2.2) <0.01 1.8
(1.3-2.3) <0.001 3.2 (2.7-3.7) <0.001 Autoantibodies (titer)
4.2 (4.0-4.4) 4.7 (4.0-5.4) NS 5.0 (4.2-5.8) NS 6.6 (5.7-7.4)
<0.001 vWF (percent) 100 (90-110) 150 (110-180) NS 160 (130-190)
<0.01 210 (180-240) <0.001 Data represent means and 95%
confidence intervals (between brackets).
[0138] TABLE-US-00009 TABLE 8 Characteristics of native LDL and of
OxLDL isolated from plasma of severe chronic renal failure patients
Native LDL OxLDL Protein/antigen ratio >100 1.1 Reactivity with
mAb-4E6 (C.sub.50 mg/dl) 25 0.02 Relative electrophoretic mobility
1 1.7 Malondialdehyde (mole/mole protein) 3 68 Substituted lysines
per apo B-100 5 130 Cholesterol/protein ratio 1.8 3.3 Free
cholesterol/cholesterol ester ratio 0.38 0.36 Phospholipid/protein
ratio 1.7 1.6 Fatty acids (%) 16:0 14 37 18:1 19 50 18:2 55 10 20:4
12 3 Data represent means of ten LDL preparations of chronic renal
failure patients. Native LDL and OxLDL patients were separated by
ion-exchange chromatography.
[0139] TABLE-US-00010 TABLE 9 Multiple regression analysis of the
dependence of OxLDL on the extent of renal failure Variable F-value
p-value Age 1.2 0.28 Sex 1.4 0.25 Hypertension 1.1 0.31
Triglycerides 1.5 0.23 HDL cholesterol 1.7 0.20 LDL cholesterol
0.99 0.32 Renal failure 14 0.0004 The data set contained 27
controls, 20 MCRF patients and 21 SCRF patients. F-values were
obtained after adjustment for the other variables. Cockcroft
creatinine clearance rates were used as a quantitative parameter
for the extent of renal failure. All linear variables were
logarithmically transformed to obtain normality for linear
regression analysis. The multiple R.sup.2 value of the multiple
regression model was 0.634.
[0140] TABLE-US-00011 TABLE 10 Multiple regression analysis of the
dependence of OxLDL on hemodialysis and LDL cholesterol levels in
severe chronic renal failure patients Variable F-value p-value Age
0.11 0.58 Sex 0.19 0.66 Hypertension 0.01 0.95 HDL cholesterol 0.02
0.89 Triglycerides 3.7 0.060 Hemodialysis 5.6 0.021 LDL cholesterol
7.1 0.0095 The data set contained 21 SCRF and 56 HEMO patients. All
linear variables were transformed logarithmically to obtain
normality for linear regression analysis. The multiple R.sup.2
value of the multiple regression model was 0.56.
[0141] TABLE-US-00012 TABLE 11 Inter-assay variation coefficients
of sandwich ELISA using 10 subsequent, independent MDA-modified LDL
preparations Inter-assay variations Concentration coefficients (nM)
(%) 10 9.6 5 7.6 2.5 8.4 1.25 13.2 0.62 12.0 0.31 13.0 0.16 12.3
0.08 15.5 0.04 16.9 0.02 13.6 0.01 11.4
DOCUMENTS
[0142] 1. Haberland M D, Fogelman A M, Edwards P A: Specificity of
receptor-mediated recognition of malondialdehyde-modified low
density lipoproteins. Proc Natl Acad Sci 1982; 79:1712-1716. [0143]
2. Holvoet P, Collen D: oxidized lipoproteins in atherosclerosis
and thrombosis. FASEB J 1994; 8:1279-8440. [0144] 3. Salonen J T,
Yla-Herttuala S, Yamamoto R, Butler S, Korpela H, Salonen R,
Nyyssonen K, Palinski W, Witztum J L: Autoantibody against oxidized
LDL and progression of carotid atherosclerosis. Lancet 339:
883-887, 1992. [0145] 4. Holvoet P, Perez G, Bernar H, Brouwers E,
Vanloo B, Rosseneu M, Collen D: Stimulation with a monoclonal
antibody (qm4E4) of scavenger receptor-mediated uptake of
chemically modified low density lipoproteins by THP-1 derived
macrophages enhances foam cell generation. J Clin Invest 93: 89-98,
1994. [0146] 5. Holvoet P, Perez G, Zhao Z, Brouwers E, Bernar H,
Collen D: Malondialdehyde-modified low density lipoproteins in
patients with atherosclerotic disease. J Clin Invest 1995;
95:2611-2619. [0147] 6. Havel R J, Eder H A, Bragdon J H: The
distribution and chemical composition of ultracentrifugally
separated lipoproteins in human sera. J Clin Invest 1955;
34:1345-1353. [0148] 7. Steinbrecher UP: Oxidation of low density
lipoprotein results in derivatization of lysine residues of
apolipoprotein B by lipid peroxide decomposition products. J Biol
Chem 1987; 262:3603-3608. [0149] 8. Sparrow C P, Partharasathy S,
Leake D S, Witztum J L, Steinberg D: Enzymatic modification of low
density lipoprotein by purified lipoxygenase plus
phospholipase-A.sub.2 mimic cell-mediated oxidative modification. J
Lipid Res 1988; 29: 745-753. [0150] 9. Holvoet P, Donck J,
Landeloos M, Brouwers E, Luijtens K, Arnout J, Lesaffre E,
Vanrenterghem Y, Collen D: Correlation between oxidized low density
lipoproteins and von Willebrand factor in chronic renal failure.
Thromb Haemostas 1996; 76:663-669. [0151] 10. Libby P, Salomon R N,
Payne D D, Schoen F J, Pober JS: Functions of vascular wall cells
related to development of transplantation-associated coronary
arteriosclerosis. Transplant Proc 1989; 21:3677-3684. [0152] 11.
Hruban R H, Beschorner W E, Baumgartner W A, Augustine S M, Ren H,
Reitz B A, Hutchins G M: Accelerated arteriosclerosis in heart
transplant recipients is associated with a T-lymphocyte-mediated
endothelialitis. Am J Pathol 1990; 137:871-882. [0153] 12. Rose E
A, Smith C R, Petrossian G A, Barr M L, Reemtsma K: Humoral immune
responses after cardiac transplantation: correlation with fatal
rejection and graft atherosclerosis. Surgery 1989; 106:203-208.
[0154] 13. Tanaka H, Sukhova G K, Swanson S J, Cybulsky M I, Schoen
F J, Libby P: Endothelial and smooth muscle cells express leukocyte
adhesion molecules heterogeneously during acute rejection of rabbit
cardiac allografts. Am J Pathol 1994; 144:938-951. [0155] 14. Crisp
S J, Dunn M J, Rose M L, Barbir M, Yacoub M H: Antiendothelial
antibodies after heart transplantation: the accelerating factor in
transplant-associated coronary artery disease? J Heart Lung
Transplant 1994; 13:81-92. [0156] 15. Grattan M T, Moreno-Cabral C
E, Starnes V A, Oyer P E, Stinson E B, Shumway N E: Cytomegalovirus
infection is associated with cardiac allograft rejection and
atherosclerosis. JAMA 1989; 261:3561-3566. [0157] 16. Koskinen P,
Lemstrom K, Brugqeman C, Lautenschlager I, Hayry P: Acute
cytomegalovirus infection induces a subendothelial inflammation
(endothelialitis) in the allograft vascular wall. A possible
linkage with enhanced allograft arteriosclerosis. Am J Pathol 1994;
144:41-50. [0158] 17. Cartier R, Dagenais F, Hollmann C, Cambron H,
Buluran J: Chronic exposure to cyclosporin affects endothelial and
smooth muscle reactivity in the rat aorta. Ann Thorac Surg 1994;
58:789-794. [0159] 18. Chin J H, Azhar S. Hoffman B B: Inactivation
of endothelial derived relaxing factor by oxidized lipoproteins. J.
Clin. Invest. 1992; 89:10-18. [0160] 19. Galle J, Schollmeyer P,
Wanner C: Cyclosporin and oxidized low density lipoproteins
synergistically potentiate vasoconstriction: influence of the
endothelium. Eur Heart J 1993; 14(Suppl):111-117. [0161] 20. Tuzcu
E M, Hobbs R E, Rincon G, Bott-Silverman C, De Franco A C, Robinson
K, McCarthy P M, Stewart R W, Guyer S, Nissen S E: Occult and
frequent transmission of atherosclerotic coronary disease with
cardiac transplantation. Insights from intravascular ultrasound.
Circulation 1995; 91:1706-1713. [0162] 21. Hoff H F, O'Neill:
Lesion-derived low density lipoprotein and oxidized low density
lipoprotein share a lability for aggregation, leading to enhanced
macrophage degradation. Arterioscler Thromb 1991; 11:1209-1222.
[0163] 22. Steinbrecher U P, Lougheed M: Scavenger
receptor-independent stimulation of cholesterol esterification in
macrophages by low density lipoprotein extracted from human aortic
intima. Arterioscler Thromb 1992; 12:608-625 33. Steinberg D,
Witztum J L: Lipoproteins and atherogenesis: current concepts. J Am
Med Assoc 1990; 264:3047-3052. [0164] 23. Ross R: The pathogenesis
of atherosclerosis: a perspective for the 1990s. Nature 1993;
362:801-809. [0165] 24. Murugesan G, Chisolm G M, Fox P L: Oxidized
low density lipoprotein inhibits the migration of aortic
endothelial cells in vitro. J Cell Biol 1993; 120: 1011-1019.
[0166] 25. Chen C H, Nguyen H H, Weilbaecher D, Luo S, Gotto A M
Jr, Henry PD: Basic growth factor reverses atherosclerotic
impairment of human coronary angiogenesis-like responses in vitro.
Atherosclerosis 1995; 116: 261-268. [0167] 26. Juckett M B, Balla
J, Balla G, Jessurun J, Jacob HS, Vercellotti GM: Ferritin protects
endothelial cells from oxidized low density lipoprotein in vitro.
Am J Pathol 1995; 147:782-789. [0168] 27. Mabile L, Fitoussi G,
Periquet B, Schmitt A, Salvayre R, Negre-Salvayre A:
Alpha-Tocopherol and trolox block the early intracellular events
(TBARS and calcium rises) elicited by oxidized low density
lipoproteins in cultured endothelial cells. Free Radic Biol Med
1995; 19:177-187. [0169] 28. Steinberg D: Clinical trials of
antioxidants in atherosclerosis: are we doing the right thing?
Lancet 1995; 346:36-38. [0170] 29. Degoulet P, Legrain M, Reach I,
Aime F, Devries C, Rojas P, Jacobs C: Mortality risk factors in
patients treated by chronic hemodialysis. Nephron 31: 103-110,
1982. [0171] 30. Neff M S, Eiser A R, Slifkin R F, Baum M, Baez A,
Gupta S, Amarga E: Patients surviving 10 years of hemodialysis. Am
J Med 74: 996-1004, 1983. [0172] 31. Cockcroft D W, Gault M H:
Prediction of creatinine clearance from serum creatinine. Nephron
16: 31-41, 1976. [0173] 32. Reade V, Tailleux A, Reade R, Harduin
P, Cachera C, Tacquet A, Fruchart J C, Fievet C: Expression of
apolipoprotein B epitopes in low density lipoproteins of
hemodialyzed patients. Kidney Int 44: 1360-1365, 1993. [0174] 33.
Sutherland W H, Walker R J, Ball M J, Stapley S A, Robertson M C:
Oxidation of low density lipoproteins from patients with renal
failure or renal transplants. Kidney Int 48: 227-236, 1995. [0175]
34. Schulz T, chiffl H, Scheithe R, Hrboticky N, Lorenz R:
Preserved antioxidative defense of lipoproteins in renal failure
and during hemodialysis. Am J Kidney Dis 25: 564-571, 1995. [0176]
35. Keane W F, Mulcahy W S, Kasiske B L, Kim Y, O'Donnell M P:
Hyperlipidemia and progressive renal disease. Kidney Int Suppl 39:
S41-S48, 1991. [0177] 36. Trachtman H. Schwob N, Maesaka J,
Valderrama E: Dietary supplementation ameliorates renal injury in
chronic puromycin aminonucleoside nephropathy. J Am Soc Nephrol 5:
1811-1819, 1995. [0178] 37. Kaplan R, Aynedjian H S, Schlondorff D,
Bank N: Renal vasoconstriction caused by short-term cholesterol
feeding is corrected by thromboxane antagonist or probucol. J Clin
Invest 86: 1707-1714, 1990. [0179] 38. Galle J, Bengen J,
Schollmeyer P, Wanner C: Oxidized lipoprotein (a) inhibits
endothelium-dependent dilation: prevention by high density
lipoprotein. Eur J Pharmacol 265: 111-115, 1994. [0180] 39.
Friedman J A, Dwyer JT: Hyperhomocysteinemia as a risk factor for
cardiovascular disease in patients undergoing hemodialysis. Nutr
Rev 53: 197-201, 1995. [0181] 40. McCully K S: Chemical pathology
of homocysteine. I. Atherogenesis. Ann Clin Lab Sci 23: 477-493,
1993. [0182] 41. Rasmussen 0, Thomsen C, Ingerslev J, Hermansen K:
Decrease of von Willebrand factor levels after a
high-monounsaturated fat diet in non-insulin-dependent diabetic
subjects. Metabolism 43: 1406-1409, 1994. [0183] 42. Reverter J C,
Escolar G, Sanz C, Cases A, Villamor N, Nieuwenhuis H K, Lopez J,
Ordinas A: Platelet activation during hemodialysis measured through
exposure of P-selectin: analysis by flow cytometric and
ultrastructural techniques. J Lab Clin Med 124: 79-85, 1994. [0184]
43. Zwaging a J J, Koomans H A; Sixma J J, Rabelink T J: Thrombus
formation and platelet-vessel wall interaction in the nephrotic
syndrome under flow conditions. J Clin Invest 93: 204-211, 1994.
[0185] 44. Zhao B, Dierichs R, Harrachruprecht B, Winterhorff H:
Oxidized LDL induces serotonin release from blood platelets. Am J
Hematol 48: 285-287, 1995. [0186] 45. Pocock S J: Subgroup
analysis, in Pocock S J (ed): Clinical trial. A practical approach.
Wiley J & Sons, Chichester, 1993, p 211-218.
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